The present invention relates to a method of detection in vitro of a target substance, notably a nucleic sequence but more generally any type of substance, in a sample. The search for target substances, notably for nucleic acid sequences, represents a primary object in numerous research laboratories implicated in numerous fields of activity, and principally in the medical or agribusiness fields. In these fields, the search for target sequences is directed for example to:                The diagnosis of a virus at the origin of diseases, such as AIDS (HIV) or hepatitis B (HBV).        The specific diagnosis of diseases of bacterial origin, such as tuberculosis or leprosy.        The diagnosis of mutations at the origin of genetic diseases or of cellular cancers.        The diagnosis of bacterial contamination in an agribusiness food chain.        The search for microorganisms implicated in the biological corrosion of pipes or of containers used in industrial processes.        
The major difficulty of the diagnosis methods used in the prior art resides in the specificity, the sensitivity, the speed and the reproducibility of the detection test used. These difficulties generally come from the nature of the labeling used. In effect, the nature of the labeling of a substance is the decisive factor in any subsequent detection permitting the following or the quantifying of said substance. Regardless of whether it concerns a human, animal or vegetable diagnosis, in agribusiness, therapy, pharmacology, research, in varied industrial processes etc., it is necessary to detect, to follow and to specifically qualify one or several target substances. In order for this detection to be optimal, it is necessary to set up high performance and sensitive labeling techniques
One of the specific techniques for labeling nucleic acids uses PCR amplification. The labeling of primers which can be used in PCR can be carried out in two ways, either by labeling of the primers, preferably at their 5′ ends or by internal marking of the amplified fragment.
The first type of labeling has the disadvantage of having a low specific activity and consequently, limits the sensitivity of the later revelation. It is possible to fix a radioactive phosphate (32P) at the 5′end of the primers. There will be one (32P) per primer. If biotin or a fluorochrome is fixed, it is possible to have at the most 3 to 4 labels per primer molecule.
If the radioactive nucleotides are incorporated in the amplicon, the specific activity is certainly more important, but it is necessary to manipulate radioactivity more. The current tendency is to replace the isotopic labeling methods with cold labeling (fluorophore, digoxigenine, biotin).
The fluorophores are sensitive to environmental changes: variations in the experimental conditions (pH, presence of oxidizing elements, etc . . . ) can displace the emission wavelength. In addition, the phenomena of fluorescence extinction (or quenching) have largely been described. The incorporation of nucleotides labeled with a fluorophore or with digoxigenine or with biotin by polymerases is of low effectiveness because these nucleotides have a strong steric hindrance which disturbs the PCR polymerization reaction.
The radioactive labeling of proteins can be carried out by using amino acids labeled with an isotope, which implicates the manipulation of radioactivity. The labeling of proteins by an antigen/antibody reaction may for its part not be so sensitive.